The broad objective of the research proposed here is to define the molecular mechanisms that regulate oppositional functions of the bHLH transcription factor Olig2 on proliferation and differentiation of neural progenitor cells. In preliminary studies, we have identified a cluster of three serine residues (Triple-S motif) in the amino terminus of Olig2 that are phosphorylated in cycling neural progenitors, but not in differentiated progeny. Mutational analysis indicates that phosphorylation of the Olig2 Triple-S motif is required for self-renewal of neural stem cells but is not required for Olig2-dependent specification of immature oligodendrocytes. The work proposed here builds upon these preliminary observations. We have five Specific Aims: Aim One is to identify the protein kinase(s) that phosphorylate Olig2 using small molecule kinase inhibitors and a kinome-wide ShRNAi library. Aim Two is to define Olig2 protein-protein interactions that are regulated by phosphorylation using TAP- Tagged wild type, phospho-null and phosphomimetic Olig2 proteins. Aim Three is to define the transcriptional functions of Olig2 phosphorylation to dictate the decision of neural progenitors to self renew or exit the cell cycle and differentiate. We will use ChIP/Seq protocols to identify direct genetic targets of phosphorylated Olig2. Aim Four is to determine a possible requirement for Olig2 Triple-S phosphorylation in embryonic patterning of spinal cord and in motor neuron development using a novel, bifunctional Olig2-tva-cre transgenic mouse neural tube explant system. Aim Five is to determine a possible requirement for Olig2 phosphorylation during oligodendrocyte maturation and CNS tumorgenesis in vivo. Orthotopic grafting studies will determine if Olig2 phosphorylation is essential for oligodendrocyte myelination, or alternatively, tumorgenesis in a mouse model of malignant glioma.